T cell lineage commitment is known to depend on a combination of transcription factors operating under the influence of Notch-Delta signaling. However, the exact roles of these factors in the molecular mechanism of lineage commitment have remained elusive. One problem is that all of the established factors themselves have roles in stem-cell maintenance and/or in other hematopoietic cell types as well as in T cells, even though the precise combination used for T-cell specification may be unique. Another problem is the complexity of the choices that are excluded during lineage commitment. T-cell precursors appear to retain access to the very distinct pathways of natural killer (NK) cell development and dendritic- cell development, possibly also to mast-cell and macrophage development, until a midpoint in their specification, and then appear to lose all these alternative potentials at once. At the same time, the cells appear to alter their self-renewal potential. It has been difficult to account for all these changes in a one- step process, especially in the absence of any known regulatory function that is clearly T-cell specific. What has been missing is a T-lineage specific regulatory factor that can be shown to control a precisely defined subset of these events. This is the role that we propose is played by Bcl11b, a recently characterized transcription factor with a highly T-lineage specific pattern of expression within the hematopoietic system. Bcl11b induction immediately precedes T-lineage commitment, and our preliminary data with a conditional Bcl11b knockout imply that this factor controls a discrete subset of lineage commitment functions, involving the repression of NK fates and stem or progenitor cell-specific maintenance pathways. In this application, we propose to test this model and to use Bcl11b to clarify regulatory linkages in the commitment process. The aims are: 1. To use Bcl11b deficiency to dissect component processes within the T-lineage commitment mechanism: (a) exclusion of NK and stem-cell regulatory programs as related to exclusion of myeloid, B, and other hematopoietic programs; and (b) commitment mechanisms that may distinguish 12 from 34 lineage T cells 2. To determine the regulatory links between Bcl11b and the Id/E protein ratio in early T-cell development 3. To determine the direct targets of Bcl11b and the ordered pathway through which Bcl11b action promotes T-lineage identity 4. To track T-lineage specification and commitment at the single cell level using new fluorescent knock- in reporters incorporated in the Bcl11b locus. PUBLIC HEALTH RELEVANCE: If stem cells are ever to be used therapeutically, it is vital to be able to understand and control commitment, the process by which stem-cell descendants give up their infinite growth potential and developmental plasticity in order to settle down to a defined biological role. This proposal will determine exactly how a newly recognized T-cell transcription factor, Bcl11b, may work to drive precursors from a stem-cell like state into a stable and useful T-cell identity, and the mechanisms that emerge will be applicable to controlling stem-cell based development in general.